Method for diagnosis of helicobacter pylori infection

ABSTRACT

A rapid, non-invasive breath-test method and device for diagnosing the presence or absence of  H. pylori  in a subject without administration of isotopic tracers is described. The device consists of a highly sensitive colorimetric ammonia sensor placed in contact with sampled subject breath. The sensor is measured using appropriate reflection spectroscopy instrumentation. The breath-test method consists of measuring a basal ammonia level with the device, administering non-isotopic urea and continuing measurement of the ammonia content in a plurality of consecutive breaths. Diagnostic differences in breath ammonia are identified between  H. pylori  infected and uninfected individuals.

RELATED APPLICATIONS

[0001] This application claims the benefit of the filing of U.S. Ser.No. 60/331,275 entitled “Method for Diagnosis of Helicobacter PyloriInfection” filed on Nov. 13, 2001, hereby incorporated by reference.

BACKGROUND

[0002] The present invention is directed to an effective diagnostictesting for the presence of gastric infection by the microorganismHelicobacter pylori.

[0003]Helicobacter pylori is estimated to be responsible for up to 90%of the cases of peptic ulcer disease (PUD) afflicts over 10% of the USpopulation sometime in their lifetime. Estimates for worldwideprevalence of H. pylori infections range from 300 million to overtwo-thirds of the world's population. H. pylori infection is alsoassociated with 650,000 annual cancer deaths worldwide from gastricadenocarcinoma. The US Communicable Disease Center recommends testingall patients presenting with PUD for diagnosis of H. pylori.

[0004] At present there are no test methods for H. pylori that satisfythe ideal conditions of being non-invasive, rapid, easy to administer,have low capital equipment and per-patient test costs, and capable ofbeing conducted in entirety during a clinician's office visit. Currentlypracticed approaches for H. pylori testing can be broken down intoinvasive (endoscopy required) and non-invasive procedures. Examples ofnon-invasive tests include: determination of antibodies to H. pylori inblood, serum, or saliva; detection of H. pylori antigens in stoolsamples; and functional tests for the presence of the bacterium's ureaseenzyme with isotope-labeled urea breath tests (UBT).

[0005] Although the non-invasive antibody-based tests are relativelyeasy to perform, they have not proved to be reliable in the generalpractitioner's office. Furthermore, they incur a blood draw and thecosts associated with the blood draw procedure. Additionally, antibodytests cannot provide a test-of-cure to demonstrate successful antibiotictreatment.

[0006] A variety of diagnostic procedures have evolved based onfunctional tests establishing presence of the urease enzyme produced byH. pylori. Urease, an enzyme found at high concentrations in theduodenum of infected individuals, hydrolyzes urea to ammonia (NH₃) andcarbon dioxide (CO₂). Tests for gastric urease, vide infra H. pylori,rely on measures of the hydrolytic by-products of urea. With respect tonon-invasive diagnosis, breath-based tests for expiredisotopically-labeled CO₂ liberated from ingested isotopic urea are wellknown in the literature. Graham described a breath test forCamphylobacter (Helicobacter) based on measurement of ¹³CO₂ releasedafter hydrolysis of ingested ¹³C-labeled urea (Graham, Lancet, May 23,1987, p1174-1177). Others have used the rapid production of isotopicallylabeled CO₂ from ingested ¹⁴C-urea or ¹³C-urea to determine the presenceor absence of the organism.

[0007] Measurement of ammonia production by the hydrolytic activity ofurease is the basis for the invasive CLO and rapid urease tests.However, to execute these tests, invasive endoscopy procedures arerequired. They are therefore neither simple, economical, convenient forthe patient, or executable in general clinical practices.

[0008] For a number of reasons, it has have proven difficult to makemeasurements of breath ammonia liberated as a by-product of H. pyloriurease. First, ammonia exists primarily as the ammonium ion at thephysiologic pH of blood, and at the pH of gastric juice there isessentially no free ammonia. While ammonia readily crosses the stomachand alveolar lining, ammonium ions are not readily absorbed. Thereforevery little ammonia finds its way from the stomach, traffics through thecirculatory system, and passes into expired air, consequently making itdifficult to measure.

[0009] A second major reason is the tight regulation of ammonia andammonium levels by the liver and kidneys. Prior to general circulation,blood from the gut is circulated through the hepatic portal vein to theliver. Normally, the combination of periportal urea cycle enzymes andperivenous glutamine synthetase results in almost complete removal ofNH₃ from blood flowing through the portal vein. Furthermore, at typicalblood pH levels of 7.4, ammonia that does pass into the generalcirculation will exist primarily as ammonium ions that are removed bythe kidney. This homeostatic regulatory system is therefore expected tominimize any fluctuations in circulating ammonia. Consequently, onlyminimal variations in breath ammonia would be expected either in normalindividuals or, by comparison, individuals infected with H. pylori.

[0010] The literature corroborates the difficulty measuring ammoniadirectly in breath and lack of clinical evidence differentiating H.pylori individuals based on breath measures, such that a simplediagnosis via breath analysis is not expected. Lipski (Lipsky PS et al.,Aust NZ J Med 22:311,1992) and Plevris (Plevris JN et.al., Lancet:1104)found no difference in blood ammonia concentration between H. pyloripositive and negative patients. Only by looking at ¹⁵NH₄ ⁺ excretion inurine was Jicong (Jicong W et al., J. Clin Micro. 30(1):181-4, 1992)able to demonstrate a difference between pylori positive and negativesubjects using nitrogen based assays. U.S. Pat. No. 4,947,861 suggeststhat by absorbing the water vapor from the breath prior to collecting atest sample, breath ammonia might be measured. However, he offers noevidence to demonstrate the utility of this maneuver and further, offersno teaching of its clinical utility or basis for deriving diagnoses.Similarly, Katzman (U.S. Pat. No. 6,067,989) suggests the use of nearinfrared analyzer for measuring breath changes in by-products (CO₂ &NH₃) of hydrolyzed isotopically labeled urea. Again however, Katzman'smethod does not teach diagnosis via ammonia, offering support only formeasuring the ¹³C-labeled CO₂ by-product as measured by others (GrahamDY et.al., Lancet, 1174-77, May 23, 1987).

[0011] Isotopic labeling has been critical in other breath measurementdiagnostics for several reasons. Labeling provides advantage towardssensitive and specific distinction of the labeled reporter by-productusing sophisticated instrumentation. The specific measurement of thelabel enables these assays to distinguish and quantify the ureahydrolysis product(s) in the presence of unlabled native hydrolysisproducts. For instance, as in the case of isotopic CO₂ based H. pyloribreath testing, the use of ¹⁴C-labeled urea allows specific detection ofthe ¹⁴CO₂ urea byproduct at nanomolar concentrations despite millimolarCO₂ concentrations in the basal breath.

[0012] With respect to use of labeled urea, it is important toappreciate that the hydrolytic by-products of CO₂ and NH₃ generatedwithin the gastrointestinal tract have vastly different fates within thebody. As indicated, ammonia is tightly controlled by homeostaticmechanisms regulating physiological processing and circulating levelswith little or no role for clearance by exhalation. In contrast, CO₂ hasmarkedly different regulatory processes affecting its circulatoryconcentration with its major route for clearance occurring through thelungs. Therefore, despite labeled CO₂ being measurable in breath andserving diagnostically via the UBT method, it is not to be expected thatammonia would provide a parallel alternative avenue to diagnosis, muchless be manifest in any diagnostically useful pattern in the breath.

[0013] There is a need for a simple, rapid non-invasive diagnostic testfor H. pylori, based on measuring ammonia in breath, without the use ofisotopically labeled reagent.

[0014] These and other limitations and problems of the past are solvedby the present invention.

BRIEF SUMMARY OF THE INVENTION

[0015] A breath test device and method for determining the presence ofH. pylori infection is disclosed and described including:

[0016] a) utilizing a sensing device capable of measuring ammonia atconcentrations of between 50 ppb to 5000 ppb and a means for collectingand passing a breath sample to the sensor means;

[0017] b) measuring the basal ammonia in an individual's breath over aperiod of 0.5 to 5 minutes in a continuous or semi-continuous manner;

[0018] c) comparing the individual's basal breath ammonia againstnormative population values wherein H. pylori uninfected individualsdisplay breath ammonia values above a predefined threshold and H. pyloriinfected individuals display breath ammonia values below said threshold;

[0019] d) administering a safe quantity of unlabeled urea to the subjectand analyzing the subject's breath for the appearance of excess ammoniaabove the basal level; and

[0020] e) comparing the individual's percentage change in post-ureabreath ammonia against normal population values wherein H. pyloriuninfected individuals display percentage changes below a predefinedthreshold and H. pylori infected individuals display percentage changesabove a given threshold.

[0021] Alternatively, the follow-on urea administration and percentagechange from basal measures can be utilized particularly on thoseindividuals exhibiting intermediate basal results that are notdefinitive for the subject's H. pylori status as a means to moreaccurately identify infected individuals.

[0022] The method and device described herein satisfies therefore anunmet need for a simple, rapid non-invasive diagnostic test for H.pylori, based on measuring ammonia in breath, without the use ofisotopically labeled reagent. Using highly sensitive calorimetricammonia sensor membranes, a color analysis instrument to determinechanges in the membrane's color and unlabeled urea, a subject's breathammonia is analyzed prior to and after ingesting the urea. A remarkableand unexpected pattern in the breath ammonia measures has beendiscovered which is useful as a diagnostic. In addition to theinstrument, sensor and materials, analytical methods for determining theH. pylori status of an individual without the use of isotopicallylabeled compounds is disclosed and described.

[0023] The invention will best be understood by reference to thefollowing detailed description of the preferred embodiment, taken inconjunction with the accompanying drawings. The discussion below isdescriptive, illustrative and exemplary and is not to be taken aslimiting the scope defined by any appended claims.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0024]FIG. 1A is a dissolve view of the components of the optical sensorincluding a fiber optic 1 and FC type connector housing, a dyed sensorfilm 2, a undyed membrane overcoat 3 for protection and reflectancebacking, a mechanical fit collar 4; and parts 5 slid over bulinose of FCconnector to provide a tight fitting sheath.

[0025]FIG. 1B is a schematic illustrating a fiber optic instrumentreader 6, three fiber optic ammonia gas sensors 7 inserted into agas-impermeable plug, a T-tube 8 with three ports for the subjectmouthpiece, the fiber optic sensors in plug and an exhaust port and abreath exhaust tube 9.

[0026]FIG. 1C shows a diagram indicating a representative ABT testsequence

[0027]FIG. 2A representative a bromocresol green (BCG) sensor reflectedsignal response change measured at two wavelengths as a function of stepchanges in ammonia exposure.

[0028]FIG. 2B representative a bromocresol green (BCG) sensor calculatedsignal RATIO response for two wavelengths as a function of step changesin ammonia exposure.

[0029]FIG. 2C representative a bromophenol blue (BPB) sensor reflectedsignal response change measured at two wavelengths as a function of stepchanges in ammonia exposure.

[0030]FIG. 2D representative a bromophenol blue (BPB) sensor calculatedsignal RATIO response for two wavelengths as a function of step changesin ammonia exposure.

[0031]FIG. 3A shows a plot of breath ammonia versus time during an ABTtest for an H. pylori negative subject

[0032]FIG. 3B shows a plot of breath ammonia versus time during an ABTtest for an H. pylori positive subject

[0033]FIG. 4 shows a bar chart indicating differentiation of H. pylorinegative and positive individuals based on higher BASELINE (Basal)ammonia breath measures (white bars) in negative individuals thanobserved in H. pylori negative individuals. The chart also shows lack ofdifference in absolute breath ammonia measure after administration ofUREA.

[0034]FIG. 5 shows a bar chart (log scale) indicating differentiation ofH. pylori negative and positive individuals based on a larger percentageincrease in breath ammonia measures after urea administration forpositive individuals than that observed in negative individuals.

DETAILED DESCRIPTION OF THE INVENTION

[0035] A device for sensing ammonia in the breath and a method forconducting and interpreting diagnostic measures indicating theHelicobacter pylori clinical status of the subject is disclosed anddescribed. Specifically, the device determines the H. pylori status ofan individual by measurement of their breath ammonia both before andafter oral administration of urea. Abnormally low basal breath ammoniameasures (before urea administration) are diagnostic for the presence ofH. pylori. After urea administration, increases in breath ammonia areindicative of H. pylori infection. Comparison of the relative change inbreath ammonia before and after urea administration is even morepredictive and is preferred for determining the presence or absence ofH. pylori. Preferably, the comparison of the basal ammonia measure andthe relative change in breath ammonia after urea administration are usedtogether to predict the H. pylori infection status of the individual.

[0036] In one embodiment, the ammonia-sensing device utilizes an opticalsensor that measures changes in ammonia concentration. In one aspect,the optical sensor includes a non-water soluble pH indicator dye,incorporated into an ammonia permeable solid-phase film or films (sensorfilms). Suitable dyes are chosen from a number of weak acid compoundsthat undergo sharp changes in their absorption spectra upon acid/basedissociation and include, but are not limited to, bromocresol green orbromophenol purple. The sensor films are composed of gas permeablehydrophobic polymers. In one aspect, these films may be porous. Thepolymers include Teflon® and related substituted ethylenic polymers.

[0037] Optical dyes are incorporated into sensor film by absorptionmethods using suitable solvents. Useful solvents are capable ofdissolving the optical indicator dye and of wetting the hydrophobicsensor film. Such solvents include alcohols such as methanol, ethanol,and isopropanol as well as organic solvents such as THF anddichloromethane. Many other suitable solvents are known to thosepracticed in organic chemistry. In one aspect, deposition of the dyeinto the film is accomplished by dipping the film into the indicator dyesolution followed by extensive rinsing of the membrane in water.Alternatively, the dye can be applied by spraying the solvent mixtureonto the film followed by rinsing. An additional alternative would be toincorporate the dye directly into the film during its manufacture.

[0038] Residual dye is immobilized within the pores of the film suchthat gaseous ammonia permeates through the film, acting as a base withthe indicator dye therein producing a change in the dye's spectralcharacteristic. The hydrophobic nature of the film prevents water anddissolved ions, including hydroxyl and hydronium ions, from interactingwith the incorporated dye thereby making the optical sensor sensitivepredominately to gaseous bases such as ammonia.

[0039] Ammonia induced color-change responses in the sensor film arepreferably measured using optical absorption-based spectroscopyinstruments. These instruments utilize reflectance measures of the pHdye's main absorption band. These reflection measures utilize two ormore wavelengths including the main optical absorption band of the dyeand a reference wavelength with changes unrelated to the dissociationstate of the indicator dye. Illumination wavelengths can be provided byany suitable means known to those practiced in the art of opticalinstrumentation including tungsten-halogen, xenon, or light emittingdiode lamps, among others. Detection of reflected light can be measuredusing photomultiplier tubes, PIN or other photosensitive devices andquantified using V/F, A/D or other methods well known to optical sensorpractitioners.

[0040] Sensor films can be affixed to an optical window that allows foroptical interrogation of the membrane by visual or instrument means. Inone aspect, the optical window is the face of a waveguide. In anotheraspect, the waveguide is a fiber optic that allows insertion of thesensor film into the test environment with remote placement of thedetection instrumentation. The ammonia breath-test (ABT) is mostconveniently conducted by placing the optical ammonia sensor into theproximal port of a breathing tube, as close as possible to the subject'smouth as feasible. By placing the sensor close to the subject's mouth,the test minimizes adsorption of breath ammonia by breath condensate,plastic materials in the breath tube or other materials that act as anammonia sink. Alternatively, the optical ammonia sensor may be placedclose to the subject's nose in a nasal mask.

[0041] The ABT is conducted by measuring a fasted subject's breathbefore and after administering urea. In one embodiment of the method,the subject's normal exhalation is measured continuously for a period ofup to 5 minutes to establish the individual's endogenous baseline,“basal measure”, or “basal ammonia measure”, of normally expiredammonia. Following determination of the basal ammonia measure, thesubject is given a safe amount of urea to ingest. After ingestion of theurea, the subject's breath is measured after a period suitable to allowdissolution of the urea in the gastric contents and to be acted upon byany putative urease enzyme from the H. pylori organism to establish apost-urea ammonia measure or “post-urea measure”.

[0042] Concurrent or subsequent to ingestion of the urea, the subjectcan be given materials or asked to perform physical maneuvers designedto enhance the urea hydrolysis to ammonia, ammonia release and/or itsappearance in breath. As an example, a pH modifier such as Al(OH)₃ orMg(OH)₂ can be administered to raise the stomach pH, shifting theequilibrium between ammonium and ammonia towards the latter. Othermaneuvers designed to increase breath ammonia concentrations mightinclude holding one's breath prior to exhalation or hyperventillating tochange blood acid/base chemistry.

[0043] Non-isotopically labeled urea can be orally administered in anumber of forms including capsules, liquids, sachets, or tablets. In oneaspect, the urea is given such that only urease in the stomach can acton the administered urea. In another aspect, the urea is administered ina fast dissolving gelatin capsule (for example, less than 3 minutes tocomplete dissolution) with sufficient water to dissolve the capsule inthe stomach. Twenty minutes was found adequate for dissolution,hydrolyis and subsequent appearance of ammonia on the subject's breath.This time might be minimized by delivering the urea in alternate formssuch as liquid, liquid gel caps or other means of presolubilizing theurea.

[0044] The novel diagnostic method includes using the basal ammoniameasure as a discriminator of infected versus uninfected individuals.The surprising results show H. pylori infected individuals have lowerbasal ammonia measures than uninfected individuals. Consequently, in oneembodiment, the method for determining H. pylori status compares thebasal ammonia ABT values against normative population standards.

[0045] It was also determined that after administering urea, the ammoniabreath test values changed to a greater degree in infected than innon-infected individuals. As one example, 300 mg of urea wasadministered resulting in post-urea ammonia levels of 400 ppb to 1000ppb ammonia. For this urea dose, the final post-urea ammonia ABT valuesare not diagnostic for the H. pylori status. Consequently, the methodfor determining H. pylori status compares the absolute or relativechange between the basal ammonia measure and post-urea ammonia measurefor an individual against normative general “population standards”. Thediagnostic test would utilize a combination of the basal ammonia measureand post-urea administration change (“post-urea ammonia measure”) inbreath ammonia measures to determine the H. pylori status of theindividual.

EXAMPLES

[0046] The following examples are provided to illustrate the device usedto measure breath ammonia, the method used to collect diagnostic breathammonia measurements and the analytical methods for diagnosing H. pyloriinfection using the Ammonia Breath Test (ABT).

Example 1 Preparation of Ammonia Sensors for ABT and Optical SensorInstrumentation

[0047] The following examples describe the preparation of an ammoniasensitive optical sensor useful for the direct determination of breathammonia measures.

[0048] In one embodiment, the ABT sensor composition is made from anammonia sensitive indicator dye and a solid phase, for example, a PTFEsolid phase in a film form. In one aspect, the sensor compositions areconstructed by administering ammonia-sensitive indicator dye(s) in anon-aqueous solvent to a solid-phase PTFE substrate such that the dye isdeposited on the solid phase in a form insoluble to aqueous-basedsolvents. Further, the characteristics of a PTFE film or a porousmembrane form are such that it is permeable to gaseous ammonia.

[0049] Optical sensor films for ABT were prepared by dissolving theoptical dyes bromocresol-green (BCG) or bromophenol-blue (BPB) inmethanol at a concentration of 0.75 mg/L. Other dyes, such as but notlimited to, any fluorescent dyes such as H2TFPP or other pH sensitivedyes can be used. In this example, porous 1 μm PTFE films were dippedinto the optical dye solutions for 20 seconds, although films of otherthicknesses are envisioned. Alternatively, dye concentrations of about0.25 mg/L to about 5 mg/L have been used to successfully prepare ammoniasensors with the required ammonia sensitivity for use in ABTmeasurements. After the film has been thoroughly wetted with the dyesolution, the film is removed from the solution, blotted dry then washedextensively with deionized water. The washed sensor films were dried andstored in the dark.

[0050] Optical sensors 1 were prepared by placing a small piece of thebromocresol-green or bromophenol-blue dyed optical sensor film over theend of a 250 μm fiber optic potted in an FC-optical connector housing.The sensor films were then mechanically fixed in place by putting on anoverlay of a second piece of undyed PTFE film around which a tightfitting collar was fitted such that the two PTFE films are held tightlyagainst the nose of the optical connector as shown in FIG. 1a. Althoughin this example the attachment was mechanical, other attachmentmechanisms are envisioned. Useful examples include the use of pH neutraladhesives or thermal bonding of the membranes to the optical fiber orwaveguide. Alternatively, the optical dye can be dissolved or suspendeddirectly in suitable castable polymers such as or polymer solutionswhich are then applied to an appropriate optical element including fiberoptics, planar waveguides, glass slides or reflective surfaces.

[0051] For H. pylori testing of individuals, three sensors (two BPB andone BCG) were inserted into the lumen of a 3-way breathing tube 8, “aT-tube”, via the side port. The breath T-tube was also fitted with adisposable mouthpiece. FIG. 1b shows an exemplary breath test sensingdevice.

[0052] The optical sensor films may be placed on a transmissive planarsurface and measured by diffuse reflectance spectroscopy or, usingsuitable optics, by transmission spectroscopy. Similarly, by adheringthe ammonia sensitive film to a transmissive planar waveguide, changesin the optical properties of the film may be measured using totalinternal reflection methods. In another aspect, the ammonia sensitivemembrane may be placed on a reflective surface and changes in the film'sabsorbance spectra measured by conventional reflection spectroscopymethods. Similarly, fluorescent dyes sensitive to ammonia could be usedto measure changes in breath ammonia.

[0053] It is recognized that other alternate means of measuring ammoniamay be available which can measure ammonia on a subject's breathincluding but not limited to electrochemical sensors, mass spectroscopy,and dye coated silica. To practice the methods described in thisinvention, the measuring device should provide ammonia sensitivity inthe range of 0.05 ppm to 5 ppm. Further, the test measuring device mustbe able to measure in the presence of water vapor, volatile organicsincluding acetone, with temperature sensitivity coefficients less than10% of scale.

[0054] Instrumentation for Optical Measurement of Ammonia SensorResponses

[0055] The instrument and typical manner of measuring the optical sensorusing the instrument is described. Changes in the optical absorbance ofthe ABT sensor described above were analyzed using solid state opticalmeasurement instruments.

[0056] In one embodiment, the measuring instrument is a multi-functionalfiber optic sensing system consisting of 3 separate optoelectronicmodules, for example, to measure 3 sensors at one time, and controlsoftware running on an attached PC. Each module contains two LED's withcenter wavelength outputs roughly matched to the sensor dye's mainabsorption band and its isobestic point, hereafter referred to as theSignal Channel and Reference Channels respectively. Modules formeasuring BCG sensors utilized LED's with center wavelengths of 620 nmand 470 nm (Hewlett Packard). Modules for measuring BPB sensors utilized600 nm and 470 nm LED's (Hewlett Packard). Alternatively, the Referencewavelength can be centered in the “Acid Absorption band” (e.g. 430-450nm for BCG) or a non-absorbing region of the optical dye's spectra (i.e.greater than 700 nm). The LEDs' outputs are coupled into an opticalcable with an FC-connector at the distal end to which the ammonia sensoris attached. The instrument alternately activates the LED's transmittingat the two separate wavelengths. The light passes through the sensor tipand returns to the instrument photodetectors after diffusely reflectingoff the film. The instrument measures changes in the absorption spectrumof the ammonia sensor as it modulates in the presence of the gas. Inaddition, the instrument makes continuous reference measurements of theLED intensities and any electronic offsets in each color channel. Afternormalizing for LED intensity and offsets, the instrument calculates aratio of the Signal wavelength (i.e. 600 nm or 620 nm) divided by theReference wavelength intensity (i.e. 470 nm, 430 nm or 700 nm). Thewavelength specific signals and Ratio are electronically stored forlater analysis.

Example 2 Representative Optical Sensor and Instrument Responses toAmmonia

[0057] To establish the sensor responses to ammonia, a BCG and BPBsensor were connected to appropriate modules and then exposed to 0 ppm,1 ppm, 4 ppm, 6 ppm and 200 ppm of ammonia in water saturated air. The200 ppm sample saturates the BPB and BCG dye response ranges and wasincluded only to show a full-range response. The individual channelsignal levels at the two wavelengths were recorded. Representativeoptical signal and Ratio plots for these two sensors are shown in FIGS.2a-d. As predicted from the BPB and BCG pK's, the BPB sensordemonstrates more of its responsivity in the 0-1 ppm range than the BCGsensor that shows a more extended response over the range of 0-6 ppm.

Example 3 Representative H. pylori Positive and Negative Subject AmmoniaBreath Test Optical Sensor Responses

[0058] Thirteen volunteers were tested for the presence of H. pyloriinfection using conventional ¹⁴C-urea breath test diagnostic procedures(Ballard Medical, Draper, Utah) in order to classify their clinicalstatus based on current medical practice. Current practice requiressubjects fast overnight (typically 8-14 hours) prior to ingesting the¹⁴C-urea capsule and subsequent collection of the subject's breath. Asimilar fasting regimen was used for the ammonia breath test (ABT). Thebreath samples were analyzed for the presence of ¹⁴C using ascintillation counter. A positive urea breath test was defined as breath¹⁴CO₂ excretion greater than 200 dpm, an indeterminate test as breath¹⁴CO₂ excretion of 50-200 dpm, and a negative test as ¹⁴CO₂ excretionless than 50 dpm. Five subjects were found positive for H. pylori andeight were identified as negative for the organism as measured by thismethod. One H. pylori positive subject (identified as S3 pre-treatmentand S14 post-treatment) was tested before and after antibiotictreatment.

[0059] To measure the subjects' breath directly with the ABT opticalsensor method, the fiber optic ammonia sensors (held at 100% RH/roomtemperature) were connected up to the fiber optic reader, inserted intothe T-tube and monitored for at least about 5 minutes in air. At the endof the air reference measurement, just prior to initiating subjectbreathing, the data files were annotated with an “AIR” event marker, asa control procedure. Subjects were then asked to breathe normally intothe device for about 5 minutes to obtain their basal endogenous breathammonia measurement, “basal ammonia measure”. At the end of the period,the data file was annotated with a “BASELINE” event marker.

[0060] Within about 1 minute of the end of the baseline e.g. basalmeasurement period, each subject was given a 300 mg capsule of unlabeledurea to ingest with 30-40 mL water. This amount of urea was deemed lowrisk in terms of undesirably affecting study volunteers. Potentiallymuch larger quantities of urea could safely be consumed by individualsfor testing purposes. As gelatin capsules were the route ofadministration utilized for this example, it is recognized that it takesseveral minutes for the ingested capsule to dissolve in the stomach,release the urea, and achieve dispersion. This factors into thesubsequent reported time-course of the subject ammonia-response to urea,“post-urea ammonia measure”; and is therefore reflected in thesubsequent definition of the ABT method. It is to be further appreciatedthat not only the amount but the manner of urea ingestion can bemodified which could influence the test time-course. For instance,consumption in liquid form as pre-dissolved urea would be expected toreduce the subject response time. Such modifications are anticipated asoptimization of the ABT method.

[0061] Following ingestion of the unlabeled urea capsule, in thisexample immediately after ingestion of the unlabeled urea capsule,subjects resumed breathing into the T-tube sensor device for 16-20minutes. The end of this data collection time period was annotated witha “UREA” file-event marker. Finally, to assess the affect of a pHmodifier, subjects were given 25-30 mL of liquid Mylanta™ antacid toraise the gastric pH and release accumulated ammonium ions in thestomach (total active ingredients of ˜2.2 g aluminum hydroxide and 2.2 gmagnesium hydroxide). Subjects began breathing into the device for afinal 20 minutes and the end of the period, “post-antacid period”, wasannotated in the data file with an “ANTACID” event marker, indicatingthe subject's “post-antacid measure”.

[0062] The signal ratio was a reliable measure of the ammonia sensorresponse. A representative plot of the sensor ratio for a H. pylorinegative subject is shown in FIG. 3a. A representative plot of thesensor ratio for a H. pylori positive subject is shown in FIG. 3b. Inthese figures, the “AIR”, “BASELINE”, “UREA”, and “ANTACID” (forexample, Mylanta) test periods are marked by labels that indicate theend of each period.

[0063] Comparison of trend plots for H. pylori positive and negativesubjects showed two distinctive trends. Those subjects negative for H.pylori by the ¹⁴C-urea breath test showed: (i) a rapid ammonia signalrise during the baseline period (about 5 minutes following the AIRperiod) and (ii) minimal change in the sensor response after ingestionof the urea capsule. In contrast, subjects positive for H. pylorishowed: (i) a remarkably flat baseline period response, followed by (ii)a marked rise in the ammonia signal after administration of the ureacapsule. Note the lack of a sharp increase in the baseline response. Themarked response to the urea capsule is especially prominent incomparison to the low baseline response of these H. pylori infectedsubjects.

Example 4 Ammonia Breath Measures for H. pylori Positive and NegativeSubjects

[0064] The following example teaches the use of the baseline (basal)ammonia measure to determine an individual's H. pylori status.

[0065] The signal Ratio data collected for the thirteen volunteers wasconverted to an absolute ammonia measure using a post-test calibration.Calibrants were prepared from pH adjusted phosphate buffered ammoniumchloride solutions. By using the equilibrium ammonia gas concentrationpredicted from Henderson-Hasselbach, water saturated calibrant ammoniagasses were prepared. The predicted headspace ammonia gas wascorroborated using an Orion ion selective electrode.

[0066] Sensors were exposed to several headspace gas buffers and allowedto equilibrate for 20 minutes. Using the final Ratio value attained inthese calibration solutions, calibration coefficients were calculatedfor each sensor. The continuous Ratio values for each sensor recordedduring the breath test were then converted to ammonia concentrations(ppm) using these calibration coefficients. Finally, the averageabsolute ammonia measure indicated by the three sensors was computed fortabulation and correlation to H. pylori status.

[0067] Although this example demonstrates the use of calibrants tocalculate the ammonia concentration of a subject's breath, any methodthat allows nomalization of sensor responses to a standard are equallyuseful and diagnostic.

[0068] The average of the two BPB and one BCG sensors' calculatedammonia for each subject (no excluded sensors or data points) is shownin Table 1. TABLE 1 Calculated ammonia values for test subjects 1-14Subject Status dpm^(†) Air Baseline Urea Antacid S1 Neg 9 0.000 0.9660.822 1.002 S2 Neg 42 0.000 0.752 0.796 0.861 S3 Pos 817 0.000 −0.0780.595 0.878 S4 Pos 2030 0.000 0.050 0.795 1.098 S5 Pos 1969 0.000 0.1740.520 0.595 S6 Neg 27 0.000 0.465 0.550 0.659 S7 Neg 10 0.000 0.2620.402 0.410 S8 Pos 922 0.000 0.022 0.076 0.128 S9 Neg 0 0.000 0.3610.557 0.625 S10 neg 22 0.000 0.411 0.815 0.866 S11 Neg 4 0.000 0.2230.360 0.285 S12 Neg 0 0.000 0.545 0.749 0.734 S13 Pos 1375 0.000 0.0220.232 0.704 S14* Neg 3 0.000 0.417 0.504 0.935

[0069] There was a wide range of breath ammonia values in the baselineperiod for H. pylori negative subjects, ranging from about 0.97 ppm toabout 0.22 ppm. Remarkably, and surprising, all H. pylori positivesubjects had lower average basal ammonia levels than H. pylori negativesubjects did. The average absolute basal ammonia measure wassignificantly lower among H. pylori positive subjects as compared to H.pylori negative subjects (0.04 ppm vs. 0.49 ppm, p=0.002). In contrast,there was no significant difference between the two groups in theirpost-urea ammonia measures (0.44 ppm vs. 0.62 ppm respectively, p=0.19)or post-antacid ammonia measures (0.68 ppm vs. 0.71 ppm respectively,p=0.86).

[0070]FIG. 4 shows the breath ammonia data from the preceding table in amanner showing the basis for differentiating infection status based onthe measured basal ammonia. The graph shows that a large group ofuninfected individuals can be differentiated from infected individualsbased on the former's higher basal ammonia measures. Several individuals(S7, S11, and S5) had intermediate basal breath ammonia measures andwould require further analysis of their urea or antacid test result todifferentiate their status. It is also significant to note that 2 weeksafter completing antibiotic treatment, subject S3 (retested as S14)demonstrated a reversion in their basal ammonia breath test value fromessentially no ammonia to over 0.4 ppm, well above the intermediatelevel.

[0071] Surprisingly, the breath ammonia measure alone afteradministering urea was not diagnostic of H. pylori infection status. Itcan be anticipated that increasing the dosage of urea may have increasedthe ammonia breath level of this group sufficiently to differentiatepositive and negative individuals. Similarly, increasing the measurementtime to allow for greater urease hydrolysis of the urea might also beused to increase the ammonia breath levels.

[0072] Percent Change Urea/Baseline

[0073] The ammonia measures for H. pylori positive and negative subjectswere not significantly different after administering the urea capsule.The relative change in ammonia measure between the basal ammonia measureand post-urea ammonia measure and between the baseline and post-antacidammonia measures were analyzed. The percentage change between the basaland post-urea ammonia measures was calculated simply as:

%(B−U) change={^((Ammonia) ^(_(urea)) ^(−Ammonia) ^(_(baseline))⁾/Ammonia _(baseline)}×100

[0074] A similar calculation of the percent change from basal topost-antacid and from post-urea to post-antacid (i.e. % B−M and % U−Mrespectively) was made. The results of these calculations are shown inTable 2. The data has been sorted on the subject's ¹⁴C-urea breath teststatus and their basal ammonia measure. TABLE 2 % change: % change: %change: ¹⁴C UBT Post Post urea vs antacid vs antacid vs Subject Statusdpm Baseline Urea antacid baseline^(a) baseline^(b) urea^(c) S1 Neg 90.97 0.82 1.00 15% 4% 22% S2 Neg 42 0.75 0.80 0.86 6% 15% 8% S12 Neg 00.55 0.75 0.73 37% 35% 2% S6 Neg 27 0.47 0.55 0.66 18% 42% 20% S14 Neg 30.42 0.50 0.94 21% 124% 86% S10 Neg 13 0.41 0.81 0.87 98% 111% 6% S9 Neg0 0.36 0.56 0.62 55% 73% 12% S7 Neg 10 0.26 0.40 0.41 53% 56% 2% S11 Neg4 0.22 0.36 0.28 62% 28% 21% S5 Pos 1969 0.17 0.52 0.59 198% 241% 14% S4Pos 2030 0.05 0.80 1.10 1494% 2101% 38% S8 Pos 922 0.02 0.08 0.13 241%473% 68% S13 Pos 1375 0.02 0.23 0.70 945% 3073% 204% S3 Pos 817 −0.080.60 0.88 866% 1230% 48%

[0075] Table 2 indicates that the H. pylori positive subjectsdemonstrate a much higher percentage increase between their basal andpost-urea ammonia measures than the H. pylori negative subjects.Similarly, the percentage increase between the basal and post-antacidammonia measures was significantly higher for the infected versusuninfected individuals. These differences are also shown in FIG. 5.

[0076] The percentage change is particularly useful in differentiatingbetween subjects with intermediate baseline responses such as thoseexhibited by S7, S11 and S5. The high percentage change in ammoniameasure after urea ingestion allowed the H. pylori positive subject 5 tobe clearly differentiated from the H. pylori negative subjects 7 and 11.

Example 5 Use of pH Modifier to Enhance Breath Ammonia

[0077] An antacid was used to modify the gastric pH and the effect onbreath ammonia measures.

[0078] Ammonia produced by the H. pylori organisms is expected to beimmediately converted to NH₄ ⁺ ammonium ions due to the low pH ofgastric juice. Ions do not readily cross the stomach lining and as such,ammonium would be expected to accumulate in the stomach with only thesmall portion of ammonia (NH₃) in equilibrium passing through thestomach lining and into the blood. Raising the gastric juice contentswould be expected to increase the concentration of NH₃ in equilibriumwith NH₄ ⁺ ions and so raise the blood concentration of ammonia.

[0079] The stomach pH was increased by drinking 40 mL of an antacid (80mg aluminum hydroxide plus 80 mg magnesium hydroxide per mL) about 20minutes after a subject had ingested 300 mg of urea. Presumably, thisshould raise the gastric juice pH by 2-5 pH units, thereby increasingthe ammonia concentration by 100 to 10,000 fold as predicted by theHenderson-Hasselbach relationship of pH and concentration for weak acidsand bases. The average percentage increase in breath ammonia for the H.pylori positive subjects was 74% versus an increase of 20% for thenegative subjects. Although the averages were not statisticallydifferent (p=0.08) from each other, there is a strong indication thatinclusion of an antacid could be used to further differentiate the twopopulations. Either insufficient antacid was administered to effect thedesired change or an antacid with a higher pH might have released theammonia in a manner adequate to achieve a statistically differentmeasure.

Example 6 Determination of Status from Baseline Ammonia Breath Test &Urea Confirmation

[0080] In the following, a method for differentiating indeterminatebaseline breath ammonia values for H. pylori positive and negativesubjects.

[0081] Example 4 shows that 10 of 13 subject's H. pylori status can bereadily determined from their Baseline ammonia breath test value alone.Namely, H. pylori negative subjects demonstrated significantly higherbaseline ammonia measures than H. pylori positive subjects.

[0082] From the table in Example 4, H. pylori negative subjects S7 andS11 demonstrate Baseline breath ammonia measures of 0.26 ppm and 0.22ppm respectively. These values are similar to those for the H. pyloripositive subject S5 (0.17 ppm). However, as identified in Example 4, H.pylori positive subjects demonstrated a larger percentage increase intheir post-urea ammonia breath measures than the H. pylori negativesubjects, making it possible to discriminate between those subjects withbreath ammonia values judged to be indeterminate by absolute normativestandards.

[0083] No license is expressly or implicitly granted to any patent orpatent applications referred to or incorporated herein. The discussionabove is descriptive, illustrative and exemplary and is not to be takenas limiting the scope defined by any appended claims.

We claim:
 1. A method for detecting the presence or absence of H. pyloriinfection in an individual by measuring the ammonia in expiration of theindividual using an ammonia sensitive sensor having a sensitivity toammonia in the range of 50 ppb to 5000 ppb, comprising: a) exposing theammonia sensitive sensor to expiration; b) deriving a basal ammoniameasure of expired ammonia over a basal measurement period; and c)comparing at least one measure of basal ammonia selected from the groupconsisting of an absolute value measurement and a rate of changemeasurement during the basal measurement period against a normative H.pylori positive population measure and a H. pylori negative populationmeasure, wherein the H. pylori status of the individual is determined.2. The method of claim 1 further comprising: a) administering a H.pylori urease enzyme substrate following determination of the basalammonia measure; b) deriving a post-substrate ammonia measure of expiredammonia over a post-urea measurement period of about 10 minutes to about90 minutes after the administration of the substrate; and c) comparingat least one measure of expired ammonia selected from the groupconsisting of the absolute measure of the post-substrate period, theabsolute difference between the measures of the basal period andpost-substrate period, the relative change between the measures of thebasal period and post-substrate period, and, the rate of change inexpired ammonia measures during the post-substrate period, againstnormative H. pylori positive and the H. pylori negative populationmeasures, wherein the H. pylori status of the individual is determined.3. The method of claim 2 further comprising: a) administering an agentintended to increase expired ammonia either coincident or subsequent toadministration of the H. pylori urease enzyme substrate; b) deriving apost-substrate and post-agent ammonia measure of expired ammonia over apost-agent measurement period of about 10 minutes to about 90 minutesafter ingestion of the substrate and agent; and c) comparing at leastone measure of expired ammonia selected from the group consisting of theabsolute measure of the post-agent period, the absolute differencebetween the measure of the basal period, post-urea period and post-agentperiod, the relative change between the measures of the basal period,post-urea period, and post-agent period, and, the rate of change inexpired ammonia measures during the basal period, post-substrate period,and post-agent period, against the normative H. pylori positive and H.pylori negative population values, wherein the H. pylori status of theindividual is determined.
 4. The method of claim 3 wherein the agentintended to increase expired ammonia either coincident or subsequent toadministration of the H. pylori urease enzyme substrate is an antacid.5. The method of claim 3 wherein the agent intended to increase expiredammonia either coincident or subsequent to administration of the H.pylori urease enzyme substrate is about 2 g aluminum hydroxide and about2 g magnesium hydroxide.
 6. The method of claim 1, 2 or 3 wherein theindividual undergoing testing fasts for at least 8 hours beforeinitiation of the method.
 7. An optical sensor for expired ammoniacomprising: a solid substrate; and an ammonia sensitive indicator dyehaving measurable spectral characteristics immobilized in or on thesolid substrate so that exposure of the dye to expired ammonia causes achange in the spectral characteristics of the ammonia-sensitiveindicator dye.
 8. The sensor of claim 7 wherein the substrate ispolytetrafluorethylene.
 9. The sensor of claim 7 wherein the indicatordye is a non-water soluble pH indicator dye.
 10. The sensor of claim 7wherein the substrate is an ammonia permeable solid-phase film.
 11. Thesensor of claim 9 wherein the indicator dye is a weak acid compound thatundergoes changes in its absorption spectra upon acid/base dissociation.12. The sensor of claim 11 wherein the weak acid compound is selectedfrom the group consisting of bromocresol green and bromophenol purple.13. The sensor of claim 7 wherein the substrate is a gas permeablehydrophobic polymer.
 14. The sensor of claim 7 wherein the substrate isporous.
 15. The sensor of claim 13 wherein the hydrophobic polymer is asubstituted ethylenic polymer.